Signaling pathway components such as Ihh/Pthrp, TGF, BMPs, Wnt/-catenin, FGFs, and Sox-related proteins represent important regulators of cartilage formation and development. These same signaling pathways and related molecules are being targeted for clinical treatment of cartilage injuries and diseases (osteoarthritis, rheumatoid arthritis, and fracture repair) and cartilage tissue engineering applications (maintenance and manipulation of mesenchymal progenitor cells (MPCs)). We provide original, unpublished data indicating that the Notch signaling pathway is another important regulator of MPC differentiation and chondrocyte maturation, leading to questions regarding the mechanisms by which Notch controls these processes. To begin addressing these questions, we propose to test the novel hypothesis that RBPj?-dependent Notch signaling suppresses MPC differentiation and chondrogenesis by interacting with stabilized -catenin to regulate Hes1 and ultimately Sox9, and is later required to promote chondrocyte maturation via cartilage specific regulation of Hes1 and Runx2 activities. To test these hypotheses, we will address two Specific Aims. Experiments in Specific Aim 1 will investigate whether Hes1 is required for MPC differentiation and chondrogenesis or the Notch-mediated suppression of MPC differentiation using limb mesenchyme specific conditional loss-of-function and genetic rescue mouse models. Secondly, we will determine whether the Wnt/beta-catenin signaling pathway is necessary and sufficient for Notch mediated induction of Hes1 and suppression of MPC differentiation. Finally, we will determine whether Hes1 suppresses MPC differentiation by directly regulating Sox9 expression. Specific Aim 2 will first examine the potential roles for both RBPJ?- dependent and -independent Notch signaling in promoting chondrocyte maturation using various tissue specific Notch gain- and loss-of-function mouse models. Additionally, we will perform in vitro experiments using primary chondrocyte cultures to identify RBPJ?-dependent Notch targets that regulate chondrocyte maturation and examine whether Notch mediates Wnt/beta-catenin or BMP signaling during this process. Secondly, we will use two different conditional Hes1 mutant mouse models to determine whether Hes1 is the primary RBPJk-dependent Notch regulator of chondrocyte maturation. Finally, in vitro studies will be performed to determine whether Hes1 promotes Runx2 activity and chondrocyte maturation via competitive interactions with the mutual co-repressor, Groucho related gene (Grg1). Completion of these aims will identify the Notch signaling mechanisms important in regulating MPC maintenance and expansion, as well as, chondrocyte maturation. These molecules will likely serve as therapeutic targets for cartilage injuries or diseases and provide us with potential tools for use in cartilage tissue engineering applications. PUBLIC HEALTH RELEVANCE: We have identified the RBPJ?-dependent Notch pathway as an important regulator of MPC differentiation and chondrocyte maturation. Our proposal will determine the exact Notch signaling mechanisms responsible for suppressing chondrogenic commitment from mesenchymal progenitor cells (MPCs) and for promoting chondrocyte maturation. Data generated by this proposal will likely implicate specific Notch signaling molecules as potential therapeutic targets for cartilage related injuries and diseases, as well as, provide potential tools in MPC maintenance and expansion for use in tissue engineering applications.